IV. Clock and the islet

Whole-body deletions of clock genes demonstrate their implication in energy metabolism, but tissue-specific deletions help with our understanding of clock proteins in the biological processes specific to each tissue. For example, while whole-body deletion of Bmal1 induces hyperglycemia independent of β-cell defects, specific deletion of Bmal1 in the liver demonstrates the role of this clock protein in mitochondrial oxidative function and deletion in the pancreas, the role of this protein in glucose-stimulated insulin
secretion (GSIS). The integration at the body level of each tissue-specific role might be the result of opposite regulations.

1. Circadian misalignment and the islet clock

In isolated islets, inversion of light phases induces reversal of PER1 oscillations without changing the amplitude or the length of the period. In addition, feeding restricted to the resting phase induces a rapid 6-hour shift phase of PER1 in the islets. The circadian release of GIP (gastric inhibitory polypeptide) and GLP1 (glucagon-like peptide 1) by the intestine may participate in the entrainment of islet oscillators by feeding rhythms via the induction of the cAMP-CREB pathway. However, other mechanisms such as autocrine effects of insulin or body temperature might also control islets oscillations. Circadian misalignments induced by constant light exposure or shift-work conditions impair amplitude, phase, and synchrony of clock gene oscillations in the islet, in parallel with a loss of β-cell function. These light conditions accelerate the development of fasting hyperglycemia and islet failure in diabetes-prone HIP rats. The latter seems to be due to a loss of insulin secretion and β-cell mass induced by an increase in β-cells apoptosis. A similar acceleration in the development of diabetes is induced by a 60% high-fat diet (Aleksey Matveyenko Lecture).

Genetic alterations in clock genes lead to disturbances in energy and metabolism, which may be linked to defects in β-cell function. Decreasing Rev-erb α expression with siRNA in mouse islet cells and in the islet cell line MIN6 reduces the expression of the lipogenic genes Srebp-1c and fatty acid synthase (Fas). In contrast, the activation of Rev-erb α in the liver inhibits Srebp-1c and Fas gene expression, suggesting that Rev-erb α has tissue-specific effects on the same metabolic pathway. More precisely, the downregulation of Rev-erb α does not affect apoptosis, but decreases proliferation. This is consistent with the decrease in the expression of genes involved in islet growth and development (ie, CyclinD1, Pdx1, or Hnf4α) measured in Clock mutant mice.
In addition to its effect on cell proliferation, downregulation of Rev-erb α in mouse islet cells impairs GSIS. Impairment of GSIS is not due to a decrease in insulin expression or in insulin content, but seems to be linked to a decrease in the amount of proteins involved in exocytosis (VAMP3, MUNC18, SNAP25, and SYNTAXYN1A) (Figure 8).

Accordingly, this decrease is also observed in Clock mutant mice. GSIS from islets has the same circadian pattern as Rev-erb α expression, whatever the diet. Modulations of the Rev-erb α transcriptional activity either by heme, leptin (through the MAPK pathway), or melatonin (through the CREB pathway) lead to rapid GSIS changes. In α cells, high glucose levels reduce Rev-erb α mRNA. Decreasing Rev-erb α expression with siRNA in pancreatic islets and in a mouse-derived α-cell line, as well as invalidation of Clock, impairs glucagon secretion induced by low glucose levels. As in β cells, this impairment is linked to a decrease in the expression of several genes coding for
exocytotic proteins like MUNC18 and SYNTAXYN1A. In addition, glucose seems to control Rev-erb α mRNA via an AMPK/NAMPT/SIRT1 pathway leading to a decrease in glucagon secretion. Rev-erb α has been tested in obese mice to see whether it is of therapeutic benefit. The treatment of diet-induced obese mice with synthetic Rev-erb agonists improved glycemia and plasma lipids and decreased fat mass and body weight (Ivan Quesada Lecture).

3. Clock and oxidative stress in β cells

Invalidation of Bmal1 in the islets impairs the hyperpolarization of the mitochondria upon glucose stimulation. This leads to a decrease in the glucose-induced ATP/ADP ratio and thus to a decrease in the signal triggering insulin granule exocytosis. The
inhibition of UCP2 (mitochondrial uncoupling protein 2) rescues the impaired GSIS in Bmal1-deficient islets, suggesting that impaired GSIS is due to an increase mitochondrial uncoupling. The increase in UCP2 induced by the absence of BMAL1 may be linked to high ROS levels or transcriptional regulations by the NAD+-dependent SIRT1, SREBP1c, and PGC-1α. β cells express low levels of antioxidant proteins and thus have a very low threshold for oxidative stress. The leucine zipper NRF2 (nuclear factor [erythroid-derived 2]-like 2) controls several limiting steps of the ROS scavenging system. NRF2 binds to the antioxidant response elements (AREs) in their promoter regions. BMAL1 binds to the Nrf2 promoter, suggesting a direct regulation of this factor by the clock machinery. Accordingly, the circadian expression of NRF2 is lost in Bmal1-/- mice. Similar regulations in neurons have been involved in the ageing phenotype developed by Bmal1-/- mice. PRDX3, which is a target of BMAL1, is also a protein involved in the prevention of apoptosis induced by oxidative stress in β cells. Finally, SESTRIN2, another target of
BMAL1 and of NRF2, controls antioxidant defenses in β cells. In accordance with a primary role of dysregulated antioxidant system in circadian-disrupted β cells, the antioxidant N-acetyl cysteine rescues the impaired GSIS in Bmal1-/- islets (Vijay Yechoor Lecture).

4. Clock and type 1 diabetes

The circadian clock may control the biological processes involved in the differentiation and activity of immune cells, since the reaction of the immune system to pathogens shows a circadian variation. For example, CRY proteins are required for the regulation of inflammatory proteins by nuclear factor-κB. BMAL2 is a redundant protein of BMAL1, which forms a heterodimer with CLOCK and is expressed in the liver and SCN. The BMAL2:CLOCK dimer controls the expression of the thrombomodulin PAI1, whose circadian expression is repressed by PER:CRY. BMAL2 controls the proliferation of peripheral CD4+ T cells through the induction of interleukin 21. As nonobese diabetic mice have decreased levels of Bmal2 and Bmal2 is present in the Idd6 locus (which confers susceptibility to the spontaneous development of type 1 diabetes in the nonobese diabetic mouse), Bmal2 may be a candidate gene for type 2 diabetes (Ute-Christine Rogner Lecture).